Composition for coating substrates for biochemical analysis

Abstract

The present invention relates to a composition for coating substrates for biochemical analysis, the relative preparation process, a coating process of a substrate using said composition (CO) and an article comprising said coated substrate.

Description

[0001] “COMPOSITION FOR COATING SUBSTRATES FOR BIOCHEMICAL ANALYSIS”

[0002] The present invention relates to a composition for coating substrates for biochemical analysis, the relative preparation process, a process for coating a substrate using said composition (CO) and an article comprising said coated substrate.

[0003] STATE OF THE ART

[0004] The immobilization of biomolecules on the surface of certain substrates is a common technique mainly used in biochemical analysis -based assays, such as molecular biodiagnostic tests.

[0005] In recent years, numerous devices and techniques have been developed for effecting these tests in order to identify and quantify contaminants in certain samples such as food, water or blood. These tests are used in different fields, including agriculture, biomedical analysis, biosensors in the pharmaceutical / nutraceutical sector and in the food sector.

[0006] In particular, the immobilization of biomolecules on suitable substrates allows the creation of biosensors, i.e. devices capable of detecting and / or quantifying a specific analyte in a sample. Biosensors usually combine a biological component (i.e. immobilized biomolecules that act as a “probe” capable of capturing a specific analyte) with a physical and / or chemical detector: the biological component interacts with the analyte present in the sample and this interaction is translated into a physical and / or chemical signal by the detector.

[0007] In order to develop biochemical assays with advantageous characteristics, the immobilization of biomolecules must be optimized.

[0008] In this context, it is known that the wettability of the solid surface (intended as the capacity of a liquid of keeping contact with the solid surface due to the intermolecular interactions present when liquid and solid come into contact) plays a significant role in the immobilization and adhesion processes, in the availability of active binding sites and in the denaturation of biomolecules on the substrate surface.

[0009] The degree of wettability of a surface can be measured by the so-called contact angle (0), i.e., the angle formed by the encountering of a liquid-vapour interface with a liquidsolid interface (whose value is determined by the balance of adhesion and cohesion forces). The tendency of a drop to flatten on the surface increases as the contact angle decreases; therefore, the contact angle is an inverse measurement of the wettability of surfaces.

[0010] In other words, there is complete wettability when the contact angle approaches 0° (i.e. the liquid is completely “distended”), whereas there is poor wettability when the contact angle approaches 180°.

[0011] Surfaces with contact angles of less than 90° are usually considered hydrophilic, whereas surfaces with contact angles greater than 90° are considered hydrophobic.

[0012] In order to optimize the immobilization of biomolecules on a surface, it is important to select a suitable surface with an adequate degree of wettability considering the type of biomolecule.

[0013] In cell immobilization processes, for example, surface wettability is a crucial factor that influences cell adhesion. Both hydrophilic and hydrophobic surfaces can be exploited in different biological applications, depending on the specific objectives. In eukaryotic cells such as embryonic stem (EB) cells, for example, a hydrophobic surface represents an optimal platform for providing uniform EB cell populations and improving the differentiation efficiency of these cells.

[0014] In the immobilization processes of DNA molecules, the degree of hydrophobicity of the surface has a significant effect on the length and conformation adopted. Short-length DNA fragments are better immobilized on hydrophilic surfaces, whereas on hydrophobic surfaces the DNA has a more compact conformation. Hydrophilic surfaces are mainly used in applications that require a greater availability of binding sites, whereas hydrophobic surfaces are mostly used for applications where it is necessary to protect the DNA from degradation phenomena or to limit interactions with other molecules.

[0015] In protein immobilization processes, the impact of the molecular weight and the percentage of helix and sheet structures on the protein conformation can be modulated by the wettability of the surface. For small globular proteins for example (such as lysozymes or ribonuclease A) or large globular proteins (such as immunoglobulins) with a low content of secondary structures, the use of hydrophobic surfaces is advisable, whereas for high- molecular-weight proteins (such as albumin or fibrinogen) the use of hydrophilic surfaces is advisable.

[0016] It is therefore important to have the possibility of varying the wettability of the surface in order to have platforms that are compatible with the various potential applications.

[0017] Despite the numerous compositions and coating techniques developed in recent years, however, there is still the need for overcoming the limitations / disadvantages that characterize the prior art.

[0018] OBJECT OF THE INVENTION

[0019] The present invention relates to a composition (CO) comprising or consisting of: i. at least one orthosilicic acid ester; ii. at least one linear or branched C1-C4 alcohol; iii. water; iv. at least one polar aprotic solvent selected from acetonitrile, tetrahydrofuran, dioxane, dimethyl sulfoxide, dimethylformamide, tetramethylurea 1,3- dimethyl-2-imidazolidinone, hexamethyl-phosphoramide and mixtures thereof; v. a pH adjuster; wherein said composition (CO) has a pH ranging from 1 to 5.5, more preferably equal to 4. The present invention also relates to a process for the preparation of said composition (CO), wherein said process comprises the following steps: a) Mixing component i., component ii. and component iii to obtain a solution (S), wherein: o the weight ratio between component i. and component ii. ranges from 1 to 5, more preferably from 1 to 3.5, and is even more preferably equal to 2.25 and / or o the weight ratio between component i. and component iii. ranges from 0.25 to 0.75, and is more preferably equal to 0,5; b) Keeping the solution (S) thus obtained under stirring for a period of time ranging from 12 to 48 hours, more preferably 24 hours; c) Adding to said solution (S) a mixture (M) comprising component ii. and component iv., wherein the volume ratio between said component ii. and component iv. in said mixture (M) ranges from 0.5: 1.5 to 1.5:0.5, and is more preferably 1 : 1 and wherein said mixture (M) is added in a quantity by volume with respect to the solution (S) ranging from 1:2 to 1: 1, more preferably 1: 1.5; d) Adding component v. until reaching a pH ranging from 1 to 5.5, more preferably equal to 4 and keeping the mixture under stirring for a time range of 0.5 to 2 hours, more preferably equal to 1 hour, at a temperature ranging from 40 to 60°C, more preferably equal to 50°C.

[0020] The present invention also relates to a process for the preparation of said composition (CO), wherein said process comprises the following steps: a) Mixing component i., component ii. and component iii in order to obtain a solution (S), wherein: • the weight ratio between component ii. and component i. ranges from 1 to 5, more preferably from 1 to 3.5, and is even more preferably equal to 2.25 and / or

[0021] • the weight ratio between component iii. and component i. ranges from 0.25 to 0.75, and is more preferably equal to 0.5; b) Keeping the solution (S) thus obtained under stirring for a period of time ranging from 12 to 48 hours, more preferably 24 hours; c) Adding to said solution (S) a mixture (M) comprising component ii. and component iv., wherein the volume ratio between said component ii. and component iv. in said mixture (M) ranges from

[0022] 0.5: 1.5 to 1.5:0.5, and is more preferably equal to 1: 1 and wherein said mixture (M) is added in a quantity by volume with respect to the solution (S) ranging from 1:2 to 1: 1, and is more preferably equal to 1: 1.5; d) Adding component v. until a pH ranging from 1 to 5.5 has been reached, more preferably equal to 4, and keeping the mixture under stirring for a time interval ranging from O.to 2 hours, more preferably equal to 1 hour, at a temperature ranging from 40 to 60°C, more preferably equal to 50°C.

[0023] The present invention also relates to a coating process of a substrate for biochemical analysis comprising the following steps: a) Optionally, a pre-wash step wherein the substrate is subjected to at least one immersion wash; b) A coating step wherein the substrate is immersed in the composition (CO) described above and subsequently removed; c) A curing step wherein the substrate is left to incubate, preferably for a time range of 36 to 72 hours, even more preferably equal to approximately 48 hours. The present invention further relates to an article comprising at least one substrate coated with the composition (CO) described above.

[0024] The present invention also relates to a method for effecting biochemical analysis, wherein said method comprises: a) an immobilization step wherein a biomolecule is immobilized on the surface of a substrate coated with the composition (CO) described above; b) an exposure step wherein a sample is brought into contact with the surface of said substrate.

[0025] DETAILED DESCRIPTION OF THE INVENTION

[0026] As already specified, the present invention relates to a surface coating technique of a substrate for biosensors that allows an optimal wettability to be obtained for the specific application desired.

[0027] It has in fact been surprisingly found that the composition (CO) according to the present invention allows certain advantages to be achieved compared to the known art.

[0028] In particular, contact angle values of the coated surface lower than 80° can be obtained. More specifically, it is possible to observe a decrease in the contact angle of 20° compared to the untreated substrate.

[0029] The possibility of selecting a contact angle within such a high range is particularly advantageous for:

[0030] • optimizing the binding kinetics between the substrate and the analyte to be immobilized;

[0031] • decreasing the formation of non-specific bonds between the substrate and the analyte to be immobilized and, consequently, reducing the onset of false negatives and / or false positives in the assay (increasing its reliability);

[0032] • improving the flow dynamics of the sample to be analyzed and of the reagents on the surface of the substrate (thus increasing the efficiency of the assay); • reducing biofouling, i.e. the accumulation of biological material, such as proteins or cells, on the surface of the substrate (the accumulation of said material can in fact hinder the correct functioning of the biosensor and have a negative effect on its sensitivity of the assay);

[0033] • obtaining a homogeneous chemical functionalization of the substrate surface, with a consequent greater homogeneity of sensitivity, which in turn results in an increase in precision in the detection of target molecules, as each point of the sensitive surface is capable of interacting uniformly (and in an optimized way) with the target molecules present in the sample analyzed;

[0034] • obtaining a better morphological homogeneity of the coating applied to the substrate surface: a uniform distribution of the functional components guarantees in fact a greater reproducibility and reliability of the measurements effected, in addition to a better interaction between the sensitive surface and the sample analyzed (thus improving the sensitivity and selectivity of the biosensor);

[0035] • obtaining a high compatibility with industrial manufacturing, as the coating process is applied with efficient, highly reproducible processes compatible with large-scale production (this is thanks to the use of materials, reagents and processes that are easily scalable and integrable in the production line, such as thin film deposition, large-scale printing or lithography);

[0036] • obtaining a high compatibility with different immobilization techniques. For example, in the case of microspotter-based techniques, where small droplets are deposited on specific areas of the surface of the biosensor, the hydrophobicity of the surface allows the size and drying of the droplet deposited to be controlled (as well as obtaining greater precision in the localization of the deposition of the same). Highly wettable surfaces, on the other hand, are to be preferred with immobilization techniques and / or biochemical analyses based on channel microfluidics (as this type of surfaces favours the uniform flow of liquids inside the microfluidic channels, avoiding the formation of air bubbles and / or sample loss).

[0037] • This allows for a better management of the samples and greater efficiency in the detection of target molecules, making biosensors more sensitive, selective and suitable for diagnostic or analytical applications.

[0038] Furthermore, the present invention allows the coated substrate to be easily restored to its original state: it is in fact possible to remove the coating layer applied by simply using oxidizing solutions at relatively low concentrations. It has been observed, for example, that the coating layer applied can be removed after about one hour by using an aqueous solution of sodium hydroxide at a concentration of 1 N.

[0039] This is possible as in amorphous glass there are some chemical bonds that can be easily hydrolyzed by the oxidizing substance, with the possibility of dissolving the layer deposited in a short time. In particular, sodium hydroxide dissociates in water, releasing hydroxide ions that are responsible for breaking the oxygen bonds between the silica atoms. These properties allow for a reversible deposition process on those materials that are inert to the oxidizing agent used, with the possibility of reusing the substrate even after several coating cycles.

[0040] Another advantage of the present invention relates to its reduced environmental impact. With respect to the reagents, in fact, it should be noted that ethanol, water and acetonitrile are compatible with green chemistry, whereas the quantities of hydrochloric acid used are reduced and there is no need for using catalysts. Although tetraethyl orthosilicate (TEOS) is a toxic reagent, it should be considered that, even for productions effected on an industrial scale, its use is limited and the final product does not have the toxicity that characterizes its precursor in free form (which is almost completely converted, thus also satisfying the green chemistry requirement relating to atom economy). With respect to the process, it should be noted that it does not require a continuous or excessive supply of heat, nor catalysts. With regard to the possible impact of recycling waste from treated substrates, it should be pointed out that silica is a compound abundantly present in nature that is easily decomposable and, if dispersed in the environment, can re-enter its natural cycle.

[0041] In addition, the present invention allows the optical and physical properties of the substrate to be kept unchanged. Furthermore, the characteristics relating to the sensor capacities remain unchanged and a modest shift in the position of the easily manageable electromagnetic modes is observed.

[0042] These characteristics also allow the present invention to be compatible with different types of sensors used for carrying out biochemical analyses, both in terms of materials used (such as metals, plastics, semiconductors or hybrids) and detection techniques, such as sensors based on surface plasmon resonance (SPR), sensors based on Bloch surface waves (BSW), sensors based on photoluminescence (PL), sensors based on surface extolled Raman spectroscopy (SERS), sensors based on fluorescence or multisignal sensors (in some cases, it is also possible to improve the performance of the biosensor).

[0043] In particular, the SPR technique allows the real-time observation of binding interactions between an analyte in solution and a ligand immobilized on a biosensor. The sensor is usually made of a glass substrate (possibly covered by a semi-transparent gold fdm) coated with a functionalized polymer. SPR sensors react to changes in the refractive index to their surface, which are directly proportional to the mass bound to the biosensor. When the mass accumulates on the sensor, for example during the binding interaction between the probe and the analyte, the refractive index increases and consequently an increase in the sensor response, expressed in resonance units (RU), is observed.

[0044] In one embodiment: • the weight ratio between component i. and component ii. ranges from 1 to 5, more preferably from 1 to 3.5, and is even more preferably equal to 2.25 and / or

[0045] • the weight ratio between component i. and component iii. ranges from 0.25 to 0.75, and is more preferably equal to 0.5.

[0046] In an alternative embodiment:

[0047] • the weight ratio between component ii. and component i. ranges from 1 to 5, more preferably from 1 to 3.5, and is even more preferably equal to 2.25 and / or

[0048] • the weight ratio between component iii. and component i. ranges from 0.25 to 0.75, and is more preferably equal to 0.5.

[0049] In one embodiment, said orthosilicic acid ester is selected from tetraethyl orthosilicate, tetramethyl orthosilicate and mixtures thereof, more preferably tetraethyl orthosilicate.

[0050] In one embodiment, said linear or branched C1-C4 alcohol is selected from methanol, ethanol, propanol and its isomers, butanol and its isomers, and mixtures thereof, more preferably ethanol (even more preferably absolute ethanol).

[0051] In one embodiment, said at least one apolar aprotic solvent is acetonitrile. Acetonitrile in fact acts as a strong dilution solvent in the preparation of the composition (CO), significantly lowering the density.

[0052] In a preferred embodiment, the water included in the composition (CO) is of an ultrapure grade (Ultrapure water UPW).

[0053] In a preferred embodiment, said pH adjuster is hydrochloric acid.

[0054] In one embodiment:

[0055] • said orthosilicic acid ester is tetraethyl orthosilicate;

[0056] • said at least one linear or branched C1-C4 alcohol is ethanol;

[0057] • said at least one apolar aprotic solvent is acetonitrile;

[0058] • said pH adjuster is hydrochloric acid Even more preferably:

[0059] • the weight ratio between tetraethyl orthosilicate and ethanol is equal to 1:2.5;

[0060] • the weight ratio between tetraethyl orthosilicate and water is equal to 2: 1.

[0061] The present invention also relates to a process for the preparation of the composition (CO) described above, wherein said process comprises the following steps: a) Mixing component i., component ii. and component iii. to obtain a solution (S), wherein:

[0062] • the weight ratio between component i. and component ii. ranges from 1 to 5, more preferably from 1 to 3.5, and is even more preferably equal to 2.25 and / or

[0063] • the weight ratio between component i. and component iii. ranges from 0.25 to 0.75, and is more preferably equal to 0.5; b) Keeping the solution (S) thus obtained under stirring for a period of time ranging from 12 to 48 hours, more preferably 24 hours; c) Adding to said solution (S) a mixture (M) comprising component ii. and component iv., wherein the volume ratio between said component ii. and component iv. in said mixture (M) ranges from 0.5: 1.5 to 1.5:0.5, and is more preferably 1 : 1 and wherein said mixture (M) is added in a quantity by volume with respect to the solution (S) ranging from 1:2 to 1: 1, and is more preferably 1: 1.5; d) Adding component v. until a pH ranging from 1 to 5.5 is reached, more preferably equal to 4, and keeping the mixture under stirring for a time range of 0.5 to 2 hours, more preferably equal to 1 hour, at a temperature ranging from 40 to 60°C, more preferably equal to 50°C.

[0064] The present invention further relates to a process for the preparation of the composition (CO) described above, wherein said process comprises the following steps: a) Mixing component i., component ii. and component iii. to obtain a solution (S), wherein:

[0065] • the weight ratio between component ii. and component i. ranges from 1 to 5, more preferably from 1 to 3.5, and is even more preferably equal to 2.25 and / or

[0066] • the weight ratio between component iii. and component i. ranges from 0.25 to 0.75, and is more preferably equal to 0.5; b) Keeping the solution (S) thus obtained under stirring for a period of time ranging from 12 to 48 hours, more preferably 24 hours; c) Adding to said solution (S) a mixture (M) comprising component ii. and component iv., wherein the volume ratio between said component ii. and component iv. in said mixture (M) ranges from 0.5: 1.5 to 1.5:0.5, and is more preferably 1 : 1 and wherein said mixture (M) is added in a quantity by volume with respect to the solution (S) ranging from 1 :2 to 1: 1, more preferably 1 : 1.5; d) Adding component v. until reaching a pH ranging from 1 to 5.5, more preferably equal to 4, and keeping the mixture under stirring for a time range of 0.5 to 2 hours, more preferably equal to 1 hour, at a temperature ranging from 40 to 60°C, more preferably equal to 50°C.

[0067] The present invention also relates to a coating process of a substrate for biochemical analysis comprising the following steps: a) Optionally, a pre-wash step in which the substrate is subjected to at least one immersion wash; b) A coating step in which the substrate is immersed in the composition (CO) described above and subsequently removed; c) A curing step in which the substrate is left to incubate, preferably for a time range of 36 to 72 hours, even more preferably equal to approximately 48 hours. The pre-wash solution can comprise a polar solvent and / or an apolar solvent. Said polar solvent can be water for example or an alcohol (such as ethanol and isopropanol), whereas said apolar solvent can be acetone, for example.

[0068] The pre-wash solution can further comprise additional ingredients such as detergents (more preferably phosphate-free detergents).

[0069] In one embodiment, the pre-wash step is repeated several times (for example from 2 to 5 times), more preferably 3 times, in the same solution or in different solutions.

[0070] The pre-wash step can comprise for example, an immersion wash in water, an immersion wash in ethanol, and an immersion wash in isopropanol (said series of washes is more preferably repeated various times).

[0071] In one embodiment, before effecting the pre-wash step a), the substrate can undergo plasma treatment.

[0072] In one embodiment, during the coating step b) the substrate is immersed in the composition (CO) using a dip-coating technique. More specifically, the substrate is immersed perpendicularly (i.e. in a vertical position) into the composition (CO) (for example with the aid of a mechanical arm). The substrate is then held inside the composition (CO) for a short time in order to eliminate any local turbulence of the solution in proximity to the substrate and allow for correct interaction.

[0073] In a preferred embodiment, before carrying out the coating step b), the composition (CO) can be diluted in a dilution solvent (D) in order to adjust its evaporation rate during the curing step c) and / orthe thickness ofthe coating layer obtained at the end ofthe process. The composition (CO) can be diluted for example to values ranging from 1:0.5 to 1: 100, more preferably from 1:4 to 1: 15, and is even more preferably equal to 1: 10 (i.e. 9 mb of dilution solvent (D) is added to 1 mb of composition (CO).

[0074] The dilution solvent (D) can be represented for example by ethanol, which has proved to be particularly suitable for the dilution values indicated above. In particular, it has been seen that starting from dilutions greater than 1: 10 with ethanol, evaporation during the curing step c) is almost instantaneous.

[0075] The immersion rate of the substrate in the composition (CO) can range from 40 to 120 mm / min, more preferably from 60 to 100 mm / min, and is even more preferably equal to about 80 mm / min.

[0076] The substrate can remain immersed in the composition (CO) for a period of time ranging from 5 to 100 seconds, more preferably from 10 to 50 seconds, and is even more preferably equal to about 25 seconds.

[0077] The extraction rate of the substrate from the composition (CO) can range from 5 to 80 mm / min, more preferably from 10 to 50 mm / min, and is even more preferably equal to approximately 20 mm / min. Higher extraction-rate values can in fact lead to the formation of bubbles visible at a macroscopic and microscopic level in the coating layer due to an excessive evaporation rate of the solvent.

[0078] In one embodiment, the curing step c) is effected at a temperature ranging from 5 to 40°C, and is more preferably equal to about 20°C, and / or in a relative humidity (RH) ranging from 40 to 60, and is more preferably equal to 50.

[0079] The curing step c) can have a duration for example of at least 36 hours, more preferably ranging from 36 to 72 hours, and is even more preferably equal to about 48 hours.

[0080] In this way, it is possible to advantageously apply a TEOS layer on the substrate surface.

[0081] The thickness of the coating layer applied to the substrate can range from 1 to 50 nm, more preferably from 1 to 30 nm, even more preferably from 1 to 5 nm. In particular, the ideal thickness ranges from 2 to 3 nm (for example 2.5 nm), as with said thickness the contact angle of the surface can be regulated more efficiently.

[0082] The thickness of the coating layer depends on the various operating parameters. In particular, it has been seen that, at the same extraction rate, the thickness decreases with an increase in the dilution of the composition (CO) in the dilution solvent (D), whereas it increases with the aging time of the “mother” solution.

[0083] It has been observed however that on planar surfaces the fdm thickness does not significantly influence the contact angle obtained, said value being simply substituted by the contact angle value of the coating.

[0084] For nanostructured surfaces, on the contrary, the wettability characteristics increase with an increase in the thickness of the coating layer deposited up to the maximum value corresponding to the contact angle of the glass layer, as an effect of the reduction of the morphological inhomogeneity of the nanostructure.

[0085] On nanostructured surfaces, for example, it has been observed that coatings around 6 nm alter the contact angle by about 9-10°, whereas for greater thicknesses the angle can decrease by up to 20°.

[0086] In one embodiment, the silica layer applied by the coating has a thickness equal to or less than 10 nm, more preferably equal to or less than 5 nm, even more preferably less than 2 nm.

[0087] This invention therefore allows the thickness of the coating layer applied to be modulated below 10 nm, thus making it possible to operate in a region wherein the shift of the surface plasmon resonance shows maximum sensitivity to an increase in the thickness of the coating. This advantage is therefore particularly relevant in the case of sensor applications that exploit the plasmonic nature of the surface, as for these applications it is very important to ensure that the analyte is as close as possible to the plasmonic surface.

[0088] At the same time, the composition according to the present invention also allows a homogeneous functionalization of the substrate surface to be obtained, resulting in a greater stability in terms of sensitivity, which in turn leads to an increase in precision in the detection of target molecules. The substrates can be made of various materials known in the state of the art. They can comprise for example glass, thermoplastic polymers (such as polyethylene, polyvinyl chloride and polystyrene), polymers of a natural origin (such as cellulose and its derivatives), metals and materials used in the semiconductor industry.

[0089] The present invention further relates to an article comprising at least one substrate coated with the composition (CO) described above.

[0090] Said article can, for example, be a biosensor or any other type of device or component useful for effecting tests based on biochemical analysis. In one embodiment, said article can consist of the coated substrate, which can be used by the end-user in conjunction with various devices for carrying out tests based on biochemical analysis.

[0091] The present invention also relates to a method for effecting biochemical analysis, wherein said method comprises: a) an immobilization step wherein a biomolecule is immobilized on the surface of a substrate coated with the composition (CO) described above; b) an exposure step wherein a sample is brought into contact with the surface of said substrate.

[0092] Said immobilization step can be effected using known techniques (even directly by the end user).

[0093] Said exposure step is used on the other hand for allowing the biomolecule immobilized on the surface to interact with the analyte present in the sample.

[0094] The interaction between the analyte and the immobilized biomolecule can be translated into a physical and / or chemical signal by a suitable detector (possibly integrated in the article).

[0095] The exposure step b) of said method can be implemented for example by simple contact of the sample with the surface of said substrate or by suitable devices. The sample for example can be put in contact with the surface of said substrate by means of microfluidic-based devices.

[0096] As already indicated, the present invention mainly relates to biosensors for conducting biochemical analysis; however, it can also find application in other fields.

[0097] It can be exploited for example in analytical separation methods such as column chromatography, size-exclusion chromatography (SEC) and electrophoresis.

[0098] The present invention can also be exploited in the medical field, for example for the coating of prostheses and the formation of nanocarriers for the controlled administration of drugs (e.g. protein molecules). In particular, thin coatings of 1-2 micrometers can be obtained that are adherent, flexible and compact (e.g. through the sol -gel technique) that can be used as protection against corrosive media and for the post -functionalization of objects made of stainless steel and bioceramics (e.g., the functionalization of these materials with bioactive or antibacterial particles can induce the prevention of infections and stimulate bone formation / growth with minimal adverse biological events).

[0099] Example 1 - preparation of the composition (CO)

[0100] A solution (S) was prepared by mixing 8 g of TEOS, 18 g of ethanol and 4 g of water.

[0101] The reagents were stirred (100 RPM) at room temperature for 24 hours. A volume equal to 6 mb of a mixture of ethanol and acetonitrile (wherein the ratio by volume v / v between the two components is 1: 1) was subsequently added to 9 mb of this mixture. The solution obtained was then brought to pH 4 by adding a solution of HC1. The solution was gently stirred (100 RPM) at 50°C for 1 hour.

[0102] Example 2 - Coating of a substrate

[0103] Two samples represented by an 0.5 cm-thick glass substrate having a nanostructured surface produced by colloidal lithography and composed of Poly(methyl methacrylate) (PMMA) having a thickness of 180 nm and gold having a thickness of 150 nm were immersed in two different coating solutions, respectively: • COATING SOLUTION SAMPLE 1: the solution produced in Example 1 was diluted in ethanol by a factor equal to 1: 10 immediately before effecting the coating step;

[0104] • COATING SOLUTION SAMPLE 2: the solution produced in Example 1 was diluted in ethanol by a factor equal to 1:6 immediately before effecting the coating step.

[0105] The immersion rate in both cases was 80 mm / min, after which the surface was immersed for 25 seconds and then extracted at a rate of 20 mm / min.

[0106] The substrate surfaces were finally left for 48 hours at room temperature (20°C) and at an RH of approximately 50%.

[0107] Example 3 - Contact angle measurement

[0108] The contact angle measurements were collected using the method defined as “drop test” with a “GBX DIGIDROP contact angle meter Model MSE”, in “surface energy” mode, using deionized water. The image analysis was effected using “Windrop++” pre-set software version 4.21.01.01 GB (GBX - Z.A. des Allobroges Ibis, rue Claude Bernard - B.P. 135 - 26104 Romans CEDEX).

[0109] In particular, the contact angle of samples 1 and 2 was measured both before and after coating in order to determine the variation of said contact angle following the treatment.

[0110] The change in the contact angle obtained by deposition of the above 1: 10 dilution in the mode and conditions indicated was 9° (contact angle measured for Sample 1 before treatment = 90°, contact angle measured for Sample 1 after treatment = 81°), whereas for the 1:6 dilution the contact angle values were reduced by 15° (contact angle measured for Sample 2 before treatment = 103°; contact angle measured for Sample 2 after treatment = 88°).

[0111] An immobilized molecule, as already indicated, can benefit from deposition on a more hydrophilic surface both in terms of maintaining its function and in terms of quantity. It is appropriate for the measurement of the contact angle to be in itself an indication of the best efficiency of the immobilization process on a surface: when the contact angle decreases, for the same volume, the diameter of the drop in which the molecule deposited for immobilization is present increases and this observation allows it to be established a priori that there will be a quantitative advantage of the signal during an assay, compared to a surface with a higher contact angle. From these observations, it can therefore be established that this change on particularly hydrophobic surfaces can already lead to an advantage for the immobilization of the molecules.

[0112] Example 4 - Thickness measurement

[0113] The substrates treated according to the previous examples were analyzed to verify the thickness of the silica layer applied at the end of the coating step.

[0114] These measurements were effected using spectroscopic ellipsometry. In particular, the ellipsometric response of the substrate was measured and characterized in order to have a reference. The ellipsometric functions of the different samples with the coating were then measured and the thickness of the same was evaluated using two different models to characterize the refractive index (silica dielectric function or Cauchy dispersion model with free parameter between 1.34 and 1.57). The thickness values obtained from the two methods coincide within 3% of relative error.

[0115] The measurements effected allowed a minimum thickness of the silica layer applied at the end of the coating step equal to approximately 1.12 nm, to be determined.

Claims

CLAIMS1 . A composition (CO) consisting of: i. at least one orthosilicic acid ester; ii. at least one linear or branched C1-C4 alcohol; iii. water; iv. at least one polar aprotic solvent selected from acetonitrile, tetrahydrofuran, dioxane, dimethyl sulfoxide, dimethylformamide, tetramethylurea 1,3- dimethyl-2-imidazolidinone, hexamethylphosphoramide and mixtures thereof; vi. a pH adjuster; wherein said composition (CO) has a pH ranging from 1 to 5.5, more preferably equal to 4.

2. The composition (CO) according to claim 1, wherein:• the weight ratio between component ii. and component i. ranges from 1 to 5, more preferably from 1 to 3.5, and is even more preferably equal to 2.25 and / or• the weight ratio between component iii. and component i. ranges from 0.25 to 0.75, and is more preferably equal to 0.5.

3. The composition (CO) according to claim 1 or 2, wherein:• the weight ratio between component ii. and component i. is equal to 2.25, and / or• the weight ratio between component iii. and component i. is equal to 0.5.

4. The composition (CO) according to any of the previous claims, wherein said orthosilicic acid ester is selected from tetraethyl orthosilicate, tetramethyl orthosilicate and mixtures thereof, and is more preferably tetraethyl orthosilicate.

5. The composition (CO) according to any of the previous claims, wherein said at least one apolar aprotic solvent is acetonitrile.

6. The composition (CO) according to any of the previous claims, wherein said linear or branched C1-C4 alcohol is selected from methanol, ethanol, propanol and its isomers, butanol and its isomers, and mixtures thereof, more preferably ethanol.

7. A process for the preparation of a composition (CO) according to claims 1 -6, wherein said process comprises the following steps: a) Mixing component i., component ii. and component iii in order to obtain a solution (S), wherein:• the weight ratio between component ii. and component i. ranges from 1 to 5, more preferably from 1 to 3.5, and is even more preferably equal to 2.25 and / or• the weight ratio between component iii. and component i. ranges from 0.25 and 0.75, and is more preferably equal to 0.5; b) Keeping the solution (S) thus obtained under stirring for a period of time ranging from 12 to 48 hours, more preferably 24 hours; c) Adding to said solution (S) a mixture (M) comprising component ii. and component iv., wherein the volume ratio between said component ii. and component iv. in said mixture (M) ranges from 0.5: 1.5 to 1.5:0.5, and is more preferably equal to 1 : 1 and wherein said mixture (M) is added in a quantity by volume with respect to the solution (S) ranging from 1:2 to 1: 1, and is more preferably equal to 1 : 1.5; d) Adding component v. until a pH ranging from 1 to 5.5 has been reached, more preferably equal to 4, and keeping the mixture under stirring for a time range of 0.5 to 2 hours, more preferably equal to 1 hour, at a temperature ranging from 40 to 60°C, more preferably equal to 50°C.

8. A coating process of a substrate for biochemical analysis comprising the following steps:a) Optionally, a pre-wash step wherein the substrate is subjected to at least one immersion wash in a polar solvent and / or one immersion wash in an apolar solvent; b) A coating step wherein the substrate is immersed in a composition (CO) comprising or consisting of: i. at least one orthosilicic acid ester; ii. at least one linear or branched C 1 -C4 alcohol; iii. water; iv. at least one polar aprotic solvent selected from acetonitrile, tetrahydrofuran, dioxane, dimethyl sulfoxide, dimethylformamide, tetramethylurea 1,3- dimethyl-2-imidazolidinone, hexamethylphosphoramide and mixtures thereof; v. a pH adjuster; wherein said composition (CO) has a pH ranging from 1 to 5.5, more preferably equal to 4 and is subsequently removed; c) A curing step wherein the substrate is left to incubate, preferably for a time range of 36 to 72 hours, even more preferably equal to approximately 48 hours.

9. An article comprising at least one substrate coated by a composition (CO) according to claims 1-6.

10. A method for effecting biochemical analyses, wherein said method comprises: a) an immobilization step wherein a biomolecule is immobilized on the surface of a coated substrate by a composition (CO) comprising or consisting of: i. at least one orthosilicic acid ester; ii. at least one linear or branched C1-C4 alcohol; iii. water; iv. at least one polar aprotic solvent selected from acetonitrile, tetrahydrofuran, dioxane, dimethyl sulfoxide, dimethylformamide,tramethylurea l,3-dimethyl-2-imidazolidinone, hexamethylphosphoramide and mixtures thereof; v. a pH adjuster; wherein said composition (CO) has a pH ranging from 1 to 5.5, and is more preferably equal to 4; b) an exposure step wherein a sample is brought into contact with the surface of said substrate.